Method for determining hydrazine in carbidobar by derivatization high performance liquid chromatography

Abstract

The invention relates to a method for determining hydrazine in carbidobar by derivatization high performance liquid chromatography. Specifically, the invention relates to a method for quality control of Carbidopa as an anti-Parkinson disease drug, and especially relates to a method for determining trace impurity hydrazine in Carbidopa by using derivatization high performance liquid chromatography. In the method, octadecylsilane chemically bonded silica is used as a chromatographic column of a filling agent, and a test solution is prepared by the following steps: taking a to-be-detected test sample Carbidopa raw material medicine, putting the Carbidopa raw material medicine into a measuring flask, precisely adding water, shaking, immediately and precisely adding a diluent containing benzaldehyde and glacial acetic acid, shaking at proper time, immediately adding a proper amount of triethylamine, and uniformly stirring; and diluting to the scale by using a diluent, and uniformly shaking to obtain the test solution. The method provided by the invention can effectively and accurately determine the trace impurity hydrazine in the carbidopa bulk drug. The method provided by the invention has excellent methodological characteristics.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for quality control of the anti-Parkinson's disease drug carbidopa, and more particularly to a method for determining trace impurities such as hydrazine in carbidopa using derivatization high performance liquid chromatography. Background Technology

[0002] Carbidopa is a peripheral decarboxylase inhibitor. Its mechanism of action primarily involves inhibiting the activity of dopa decarboxylase, reducing the extracerebral conversion of levodopa to dopamine. Carbidopa does not readily enter the central nervous system, only inhibiting the peripheral conversion of levodopa to dopamine, thus increasing the circulating levodopa level and consequently increasing the amount of levodopa entering the central nervous system. This mechanism helps increase the concentration of levodopa in the brain, thereby increasing the bioavailability of dopamine and improving motor symptoms in Parkinson's disease patients. Carbidopa itself can hardly cross the blood-brain barrier, therefore it does not act directly on the central nervous system, but rather exerts its effect by enhancing the efficacy of levodopa. Clinically, carbidopa is often used in combination with levodopa for Parkinson's disease and Parkinsonian syndrome.

[0003] The chemical name of carbidopa is (S)-α-methyl-α-hydrazino-3,4-dihydroxyphenylpropionic acid, and its chemical structural formula is as follows: .

[0004] Carbidopa contains a hydrazine group (-HN-NH2) in its structure, which may degrade to hydrazine under certain conditions. For example, under acidic conditions, the protonation of the hydrazine group reduces its electron cloud density, making it susceptible to hydrolytic cleavage to generate (S)-α-methyl-3,4-dihydroxyphenylpropionic acid and hydrazine. Alternatively, an alkaline environment can accelerate the β-elimination reaction of the hydrazine group. When the pH exceeds 8.0, the α-hydrogen of the hydrazine group is removed to form a carbanion, which then cleaves to release hydrazine and generate (S)-α-methylene-3,4-dihydroxyphenylpropionic acid. Furthermore, high temperatures promote the thermal decomposition of the hydrazine group; under light or in the presence of transition metal ions, the hydrazine group is oxidized to a hydrazine radical, which further disproportionates to generate hydrazine and diimine.

[0005] Studies have shown that hydrazine compounds are alkylating agents. In vivo, their metabolic activation forms highly reactive methyl intermediates such as carbocations, carbon-centered radicals, and oxygen-centered radicals. These methyl groups react with DNA bases in vivo, forming adducts that can cause DNA damage and gene mutations. Therefore, the hydrazine content in active pharmaceutical ingredients (APIs) containing hydrazine impurities must be controlled. These hydrazine impurities may be process intermediates during API preparation or degradation impurities.

[0006] However, monitoring the content of hydrazine, an impurity in active pharmaceutical ingredients (APIs), is relatively difficult due to the following reasons: hydrazine is highly polar, has a low relative molecular mass, lacks chromophores, and has no carbon atoms; hydrazine is highly reactive, easily causing side reactions and resulting in false positives; hydrazine is strongly basic, readily interacting with silanol groups on the chromatographic column, causing peak tailing, thus requiring careful column selection; and the permissible content of hydrazine impurities in pharmaceuticals is low, typically below 50 ppm or even lower. Therefore, developing methods for determining low-impact hydrazine in APIs presents a significant challenge.

[0007] It is known that some pharmaceutical raw materials containing hydrazine impurities use benzaldehyde derivatization to generate dibenzylhydrazine, which is detectable under ultraviolet light, for trace hydrazine detection. The basic principle is as follows: .

[0008] Cui's paper (Li Cui, Kaina Jiang, David Q. Liu, et al. Simultaneous quantitation of trace level hydrazine and acetohydrazide in pharmaceuticals by benzaldehyde derivatization with sample 'matrix matching' followed by liquid chromatography-mass spectrometry. J. Chromatogr. A 1462 (2016) 73-79) discloses a method for simultaneously quantifying trace levels of hydrazine and acetohydrazide in pharmaceuticals by benzaldehyde derivatization with sample 'matrix matching' followed by liquid chromatography-mass spectrometry. In this method, a methanol-water mixture is used as the sample diluent; 0.4% benzaldehyde or 0.4% benzaldehyde / 50mM benzoic acid is used as the derivatization reagent solution (where benzoic acid is used as an enhancer for the derivatization reagent); the derivatization reagent is added to the test sample (which may contain impurities such as hydrazine or acetylhydrazine API), vortexed for about 10 seconds, allowed to stand at room temperature for 1 hour, and then determined by LC-MS. The LC is a reversed-phase high-performance liquid chromatography system with a C18 column and water / acetonitrile containing 0.1% formic acid as the mobile phase.

[0009] The European Pharmacopoeia 11th edition describes the determination of hydrazine (H2N-NH2) impurities in allopurinol using normal-phase high-performance liquid chromatography (HPLC) with an isopropanol / hexane (5:95) mobile phase. Specifically, a dilute sodium hydroxide solution / methanol mixture is used as the solvent mixture. Benzaldehyde is dissolved in the solvent mixture to prepare a derivatizing reagent. The hydrazine-containing sample is dissolved in the solvent mixture, and the derivatizing reagent is added. After standing at room temperature for 2.5 hours, hexane is added for extraction and separation. The hexane layer is used as the test solution. Detection is performed at 310 nm using a cyanosylsilane silica gel column. The United States Pharmacopeia 2024 edition also uses a similar normal-phase HPLC method to determine hydrazine impurities in allopurinol as the European Pharmacopoeia 11th edition.

[0010] In addition, the 11th edition of the European Pharmacopoeia describes the determination of the impurity hydrazine (H2N-NH2) in isoniazid using reversed-phase high-performance liquid chromatography (RP-HPLC) with an acetonitrile / water (60:40) mobile phase. Specifically, an acetonitrile / water mixture is used as the solvent mixture, benzaldehyde is dissolved in methanol to prepare a derivatizing reagent, the test sample containing the impurity hydrazine is dissolved in water, and the derivatizing reagent is added and mixed. The mixture is allowed to stand for 45 min to allow the derivatization reaction between the impurity hydrazine and benzaldehyde to completely generate benzaldehyde azine. The mixture is then diluted with the solvent mixture, and hydrazine sulfate is used as a reference standard to prepare a reference solution using the same method. The impurity hydrazine in the drug substance is detected at 30 nm using a C18 column.

[0011] However, the inventors have discovered that the methods described above for determining hydrazine impurities in other active pharmaceutical ingredients are not suitable for determining trace amounts of hydrazine impurities in carbidopa active pharmaceutical ingredient. For example, allopurinol does not produce hydrazine due to degradation; and although isoniazid theoretically has the potential to degrade, its degradation conditions and degradation kinetics are completely different from those of the carbidopa of this invention. Therefore, the methods for determining hydrazine impurities in these other active pharmaceutical ingredients cannot be directly applied to the determination of hydrazine impurities in the carbidopa of this invention.

[0012] The current edition of the Chinese Pharmacopoeia does not monitor hydrazine, an impurity in carbidopa and its preparations.

[0013] The 11th edition of the European Pharmacopoeia lists the limit for the impurity hydrazine in carbidopa raw material determined by TLC. Specifically, test solution a is prepared by dissolving the sample in dilute hydrochloric acid; after complex treatment of a strongly basic anion exchange resin, test solution a is added, followed by a mixture of salicylaldehyde (2-hydroxybenzaldehyde) / methanol / phosphate buffer; the mixture is shaken thoroughly for 1 minute and heated at 60°C for 15 minutes; after cooling, toluene is added, the mixture is shaken vigorously, centrifuged, and the toluene layer is separated to obtain test solution b; this solution is then separated on a silanized silica gel TLC plate using water / methanol (10:20) as the developing solvent and detected under 365 nm UV light. This pharmacopoeia method uses salicylaldehyde as a derivatizing reagent, has a complex sample preparation process, and can only qualitatively determine the limit of the sample, not quantitatively.

[0014] There is still a long-awaited opportunity in the field for new and effective methods to determine trace amounts of hydrazine, an impurity in carbidopa. Summary of the Invention

[0015] The purpose of this invention is to provide a novel and effective method for determining trace impurities of hydrazine in the anti-Parkinson's disease drug carbidopa, and more particularly, a method for determining trace impurities of hydrazine in carbidopa using derivatization high-performance liquid chromatography. It has been found that the method provided by this invention can effectively and stably determine trace impurities of hydrazine in carbidopa, and this invention is based on such findings.

[0016] Therefore, the first aspect of the present invention provides a method for determining hydrazine in the active pharmaceutical ingredient carbidopa, the method employing pre-column derivatization-reversed-phase high-performance liquid chromatography, comprising the following steps: (1) Provide a chromatography system: including a high performance liquid chromatograph, a chromatographic column with octadecylsilane bonded silica gel as the packing material, a mobile phase of acetonitrile-water (70:30, v / v), and a detection wavelength of 305 nm; (2) Preparation of solution: (2a) Diluent: 2% benzaldehyde solution prepared using anhydrous methanol as solvent; (2b) Test solution: Weigh 25 mg of the carbidopa raw material to be tested accurately, place it in a 10 ml volumetric flask, add 1 ml of water accurately, shake for 15 seconds, immediately add 7 ml of diluent and 100 μl of glacial acetic acid accurately, shake for 60 seconds, immediately add 100 µl to 450 µl of triethylamine (preferably 300 µl), dilute to the mark with diluent, shake well, and the test solution is obtained (carbidopa concentration is about 2500 μg / ml); (2c) Hydrazine sulfate stock solution: Weigh 20.3 mg of hydrazine sulfate reference standard accurately, place it in a 200 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and you will get (approximately equivalent to 25 μg / ml of free hydrazine); (2d) Hydrazine sulfate solution: Accurately measure 1 ml of hydrazine sulfate stock solution, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well to obtain (approximately equivalent to 0.5 μg / ml of free hydrazine); (2e) Reference solution (free hydrazine 0.05 μg / ml): Accurately measure 1 ml of hydrazine sulfate solution and prepare the reference solution (approximately equivalent to containing 0.05 μg / ml of free hydrazine) by following the same procedure as the test solution preparation method starting from "place in a 10 ml volumetric flask" (the hydrazine in this reference solution is derivatized to dibenzylhydrazine). (2f) Blank solution: Accurately measure 1 ml of water and prepare it according to the method of preparing the test solution, starting from "place in a 10 ml volumetric flask" (equivalent to a free hydrazine concentration of 0); (3) System suitability test and requirements: After the liquid chromatography system has stabilized, inject a blank solution and record the chromatogram. The blank solution should not cause any interference at the retention time of the dibenzylhydrazine peak. Inject a reference solution and record the chromatogram. The system suitability test should meet the routine requirements. (4) Determination, limit judgment, and result calculation: Inject the reference solution and the test solution into the liquid chromatograph separately, and record the chromatograms; Limit: If there is a chromatographic peak in the chromatogram of the test sample solution with the same retention time as the dibenzylhydrazine peak, compare its peak area with the main peak area of ​​the reference solution. If the dibenzylhydrazine peak area in the chromatogram of the test sample solution is not greater than the main peak area of ​​the reference solution, it means that the limit of hydrazine content in the test sample is not greater than 20 ppm. The hydrazine content is calculated using the external standard method based on the peak area of ​​dibenzylhydrazine, in ppm. The formula is as follows: Hydrazine content = Sample peak area / Reference peak area × Reference concentration / Sample weight × Sample dilution factor × Conversion factor; In the formula, Conversion factor = Relative molecular mass of hydrazine / Molecular mass of hydrazine sulfate =32.0452 / 130.12 =0.2463.

[0017] According to the method of the first aspect of the present invention, the chromatographic column in step (1) is a Welch Xtimate C18 column.

[0018] According to the method of the first aspect of the present invention, the chromatographic column in step (1) is a Welch Xtimate C18 column with an inner diameter of 4.6 mm.

[0019] According to the method of the first aspect of the present invention, the chromatographic column in step (1) is a Welch Xtimate C18 column with a length of 250 mm.

[0020] According to the method of the first aspect of the present invention, the chromatographic column in step (1) is a Welch Xtimate C18 column with a packing particle size of 5 μm.

[0021] According to the method of the first aspect of the present invention, the flow rate of the mobile phase in step (1) is 1.0 ml / min.

[0022] According to the method of the first aspect of the present invention, the temperature of the sample tray in step (1) is 10°C.

[0023] According to the method of the first aspect of the present invention, the injection volume in step (1) is 20 μl.

[0024] According to the method of the first aspect of the present invention, the test solution obtained in step (2b) is injected within 3 hours.

[0025] According to the method of the first aspect of the present invention, the test solution obtained in step (2b) is injected within 2 hours.

[0026] According to the method of the first aspect of the present invention, in step (3) system suitability test and requirements, the RSD of the peak area of ​​dibenzylhydrazine in the reference solution injected for six consecutive injections shall not be greater than 2.0%.

[0027] According to the method of the first aspect of the present invention, in step (3) system suitability test and requirements, the theoretical plate number of the dibenzylhydrazine peak in the chromatogram of the reference solution is not less than 5000.

[0028] According to the method of the first aspect of the present invention, the active pharmaceutical ingredient (API) is considered to meet the requirements when the hydrazine content in the API carbidopa is less than 20 ppm in step (4).

[0029] According to the method of the first aspect of the present invention, the active pharmaceutical ingredient (API) is considered to meet the requirements when the hydrazine content in the API carbidopa is less than 15 ppm in step (4).

[0030] According to the method of the first aspect of the present invention, the active pharmaceutical ingredient (API) is considered to meet the requirements when the hydrazine content in the API carbidopa is less than 10 ppm in step (4). Attached Figure Description

[0031] Figure 1 Chromatogram of blank solution.

[0032] Figure 2 Typical chromatogram of the reference solution.

[0033] Figure 3 Typical chromatogram of the test sample solution after 0 hours of stability testing.

[0034] Figure 4 Typical chromatogram of the test sample solution after 2 hours of stability testing.

[0035] Figure 5 Typical HPLC chromatogram of linear solution L2.

[0036] Figure 6 Typical HPLC chromatogram of linear solution L6.

[0037] Figure 7 Typical HPLC chromatogram of solution for the sensitivity test of hydrazine in carbidopa analysis.

[0038] Figure 8 Typical HPLC chromatogram of the limit of quantitation solution for sensitivity testing of hydrazine in carbidopa analysis method. Detailed Implementation

[0039] The invention will be further illustrated in detail below through specific examples. Unless otherwise specified, all materials used are readily available in the art.

[0040] The reference standard hydrazine sulfate used in this invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (purity 99.5%, batch number Z22J10Y93658). The four batches of carbidopa raw material tested were all produced by the applicant, Zhejiang Yefeng Pharmaceutical Co., Ltd.: carbidopa (validation batch, batch number 230402), carbidopa (batch number 240802), carbidopa (batch number 250101), and carbidopa (batch number 250201). In all types of tests below, unless otherwise specified, the test sample used refers to batch 250201.

[0041] The liquid chromatographs used in this invention are of two brands / models: Dionex Ultimate 3000 and Shimadzu LC-20AT; the electronic balance used in this invention is a METTLER TOLEDO XPE205; the chromatographic column used in this invention is a Welch Xtimate C18 column with dimensions of 4.6m × 250mm × 5µm; and the water used in this invention is ultrapure water prepared by an ultrapure water system Milli-Q.

[0042] The acetonitrile used in this invention was purchased from Merck's HPLC grade (batch number JB149030), and the anhydrous methanol, benzaldehyde, triethylamine, and glacial acetic acid used in this invention were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] Example 1: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment describes a method for determining hydrazine in the active pharmaceutical ingredient carbidopa, which employs pre-column derivatization-reversed-phase high-performance liquid chromatography.

[0044] (1) Provide a chromatography system: A high-performance liquid chromatograph is provided, and the following chromatographic conditions are used: a column packed with octadecylsilane-bonded silica gel (in this example, a Welch Xtimate C18 column with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm is used), a column temperature of 30 °C, a mobile phase of acetonitrile-water (70:30, v / v), a flow rate of 1.0 ml / min, a detection wavelength of 305 nm, an injection plate temperature of 10 °C, and an injection volume of 20 μl. (2) Preparation of solution: (2a) Diluent: 2% benzaldehyde solution prepared using anhydrous methanol as solvent; (2b) Test solution: Weigh approximately 25 mg of the carbidopa raw material to be tested, place it in a 10 ml volumetric flask, add 1 ml of water accurately, shake for 15 seconds, immediately add 7 ml of diluent and 100 μl of glacial acetic acid accurately, shake for 60 seconds, immediately add 300 μl of triethylamine, dilute to the mark with diluent, shake well, and the test solution is obtained (carbidopa concentration is approximately 2500 μg / ml, injection within 2 hours) (hydrazine that may be present in this test solution is derivatized to dibenzylhydrazine); (2c) Hydrazine sulfate stock solution: Weigh approximately 20.3 mg of hydrazine sulfate reference standard accurately, place it in a 200 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is obtained (approximately equivalent to 25 μg / ml of free hydrazine). (2d) Hydrazine sulfate solution (free hydrazine 0.5 μg / ml): Accurately measure 1 ml of hydrazine sulfate stock solution, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well to obtain (approximately equivalent to 0.5 μg / ml of free hydrazine); (2e) Reference solution (free hydrazine 0.05 μg / ml): Accurately measure 1 ml of hydrazine sulfate solution and prepare the reference solution (approximately equivalent to containing 0.05 μg / ml of free hydrazine) by following the same procedure as the test solution preparation method starting from "place in a 10 ml volumetric flask" (the hydrazine in this reference solution is derivatized to dibenzylhydrazine). (2f) Blank solution: Accurately measure 1 ml of water and prepare it according to the method of preparing the test solution, starting from "place in a 10 ml volumetric flask" (equivalent to a free hydrazine concentration of 0); (3) System suitability test and requirements: After the liquid chromatography system has stabilized, inject a blank solution and record the chromatogram. The blank solution should not cause any interference at the retention time of the dibenzylhydrazine peak. Inject a reference solution and record the chromatogram. The RSD of the dibenzylhydrazine peak area in the reference solution of 6 consecutive injections should not be greater than 2.0%. The theoretical plate number of the dibenzylhydrazine peak in the chromatogram of the reference solution should not be less than 5000. (4) Determination, limit judgment, and result calculation: Inject the reference solution and the test solution into the liquid chromatograph separately, and record the chromatograms; Limit: If there is a chromatographic peak in the chromatogram of the test sample solution with the same retention time as the dibenzylhydrazine peak, compare its peak area with the main peak area of ​​the reference solution. If the dibenzylhydrazine peak area in the chromatogram of the test sample solution is not greater than the main peak area of ​​the reference solution, it means that the limit of hydrazine content in the test sample is not greater than 20 ppm. The hydrazine content is calculated using the external standard method based on the peak area of ​​dibenzylhydrazine, in ppm. The formula is as follows: Hydrazine content = Sample peak area / Reference peak area × Reference concentration / Sample weight × Sample dilution factor × Conversion factor; In the formula, Conversion factor = Relative molecular mass of hydrazine / Molecular mass of hydrazine sulfate =32.0452 / 130.12 =0.2463.

[0045] According to the limits for hydrazine in carbidopa as listed in the European Pharmacopoeia, the limit for hydrazine content in carbidopa used as a pharmaceutical raw material is generally required to be less than or equal to 20 ppm.

[0046] Four batches of samples produced by the applicant, Zhejiang Yefeng Pharmaceutical Co., Ltd., were tested according to the method in Example 1 of this paper: carbidopa (validation batch, batch number 230402), carbidopa (batch number 240802), carbidopa (batch number 250101), and carbidopa (batch number 250201). The hydrazine content was calculated by the external standard method, and the results are as follows: batch 230402 was 5.3 ppm, batch 240802 was 4.8 ppm, batch 250101 was 7.3 ppm, and batch 250201 was 5.6 ppm. All of these are lower than the general limit of 20 ppm required for this product in the art (e.g., European Pharmacopoeia 11th edition), and the results are all less than 10 ppm.

[0047] Example 2: Validation of the Analytical Method This embodiment verifies the method provided in Embodiment 1.

[0048] (1) System suitability test As described in Example 1, blank solution and reference solution were injected into the liquid chromatography system and chromatograms were recorded.

[0049] The chromatogram of the blank solution is as follows: Figure 1 As shown, there is no chromatographic peak after the retention time of 6 min, which does not interfere with the determination of the dibenzylhydrazine peak at the retention time of approximately 9.5 min.

[0050] The reference solution was injected six times consecutively, and the results are summarized below:

[0051] A typical chromatogram (first injection of reference solution in a series) is as follows: Figure 2 As shown in the figure. The results indicate that the average peak area of ​​the dibenzylhydrazine peak in the chromatograms obtained from six consecutive injections of the reference solution was 0.822, the RSD was 0.38%, the theoretical plate number of the dibenzylhydrazine peak was greater than 10,000, far exceeding the 5,000 or higher typically required in this field, and the retention time of the dibenzylhydrazine peak was approximately 9.5 min. The results of the blank solution chromatogram show that the detection of dibenzylhydrazine was not interfered with. The system suitability test results meet the validation requirements.

[0052] (2) Solution stability Take the reference solution and test solution obtained from the derivatization reaction in Example 1, place them at 10°C (injection tray) for different times, and then inject them for determination.

[0053] Another hydrazine sulfate solution obtained in Example 1 was placed at room temperature for an appropriate time before undergoing a derivatization reaction and then injected. The chromatogram was recorded to examine the stability of the solution.

[0054] The following formula is used to calculate the percentage relative change of the sample after different placement times compared to time 0: , In the formula, RD% is the absolute value of the relative change (%) with respect to 0h; Xn is the peak area (content) (A) at the nth hour of injection; X0 represents the peak area (content) at injection time 0 hours (A).

[0055] For stability testing, the relative change in peak area at each time point is calculated using the formula above as time progresses for both the reference solution and the test solution.

[0056] The stability (RD%) of hydrazine sulfate solution (free hydrazine 0.5 μg / ml) after being placed at room temperature for 24 hours and 48 hours before derivatization was 1.4% and -2.5% respectively, compared to 0 hours.

[0057] The stability (RD%) of the reference solution (free hydrazine 0.05 μg / ml) after being placed at 10 °C for 4 hr, 8 hr, 24 hr, and 48 hr relative to that at 0 hr were 1.3%, -1.1%, 3.1%, and -1.3%, respectively.

[0058] The stability (RD%) of the test solution after being placed at 10℃ for 0.25hr, 0.5hr, 0.75hr, 1hr, 2hr, 3hr, 4hr, 6hr, and 9hr relative to that at 0hr was 1.5%, -3.2%, -2.6%, 2.2%, -1.7%, 3.4%, 8.8%, 13.3%, and 17.4%, respectively.

[0059] The above results indicate that: The hydrazine sulfate solution was left at room temperature for 48 hours and then prepared into a reference solution. The peak area of ​​the dibenzylhydrazine peak in the reference solution at each time point was compared with the peak area at 0 h. The absolute value of the relative deviation was less than 10.0%, especially less than 5%, indicating that the solution was stable. When the reference solution was placed at 10°C for 48 hours, the peak area of ​​the dibenzylhydrazine peak in the reference solution at each time point was compared with the peak area at 0 h. The absolute value of the relative deviation was less than 10.0%, especially less than 5%, indicating that the solution was stable. Since the hydrazine sulfate solution and the reference solution were prepared with hydrazine sulfate, there was no problem of degradation and hydrazine production, which is different from the situation where carbidopa may degrade and produce hydrazine, causing unstable test results. The test solution was placed at 10°C for 3 hours. The relative deviation of the dibenzylhydrazine peak content at each time point compared to the content at 0 h was less than 10.0%, especially less than 5.0%. However, the relative deviation approached 10% at 4 h, and exceeded 10% at 6 h and 9 h, both being positive. This indicates that the presence of carbidopa in the test solution, and the subsequent reaction of carbidopa with benzaldehyde to form dibenzylhydrazine after degradation into hydrazine, contribute to the formation of dibenzylhydrazine. From this perspective, it is meaningful to avoid or reduce the continued degradation and derivatization of carbidopa into dibenzylhydrazine in the test solution to allow more time for the determination. It is evident that the carbidopa degradation amount in the test solution obtained using step (2b) of Example 1 of this invention is limited within 3 hours, especially within 2 hours. Unexpectedly, this limited degradation was found to be related to the use of glacial acetic acid and triethylamine in the preparation of the test solution.

[0060] Therefore, the reference solution exhibits good stability after 48 hours at 10°C, the hydrazine sulfate solution exhibits good stability after 48 hours at room temperature, and the test solution exhibits good stability after 3 hours, especially 2 hours, at 10°C. The test solution should be injected within 3 hours of preparation, with injection within 2 hours being preferred. In the context of this invention, unless otherwise stated, all sample tests are performed within 2 hours.

[0061] Typical chromatograms for the stability tests of the test sample solution at 0h and 2h are shown in the figure below. Figure 3 and Figure 4 .

[0062] (3) Linearity and Range The following linear solutions were prepared using the hydrazine sulfate stock solution described in Example 1: Linear solution L1: Accurately measure 5 ml of linear solution L6, place it in a 200 ml volumetric flask, dilute with water to the mark, shake well, take 1 ml, and follow the same procedure as in Example 1, "(2b) Test solution", starting from "place in a 10 ml volumetric flask", to obtain the linear solution; Linear solution L2: Accurately measure 1 ml of linear solution L6, place it in a 10 ml volumetric flask, dilute with water to the mark, shake well, take 1 ml, and follow the same procedure as in Example 1, "(2b) Test solution", starting from "place in a 10 ml volumetric flask", to obtain the linear solution; Linear solution L3: Accurately measure 5 ml of linear solution L6, place it in a 20 ml volumetric flask, dilute with water to the mark, shake well, take 1 ml, and follow the same procedure as in Example 1, "(2b) Test solution", starting from "place in a 10 ml volumetric flask", to prepare the linear solution; Linear solution L4: Accurately measure 1 ml of the hydrazine sulfate stock solution prepared according to step (2c) of Example 1, place it in a 50 ml volumetric flask, dilute with water to the mark, shake well, take 1 ml, and follow the same procedure as in step "(2b) test solution" of Example 1, starting from "place in a 10 ml volumetric flask", to prepare a linear solution; Linear solution L5: Accurately measure 3 ml of the hydrazine sulfate stock solution prepared according to step (2c) of Example 1, place it in a 100 ml volumetric flask, dilute with water to the mark, shake well, take 1 ml, and follow the same procedure as in step "(2b) test solution" of Example 1, starting from "place in a 10 ml volumetric flask", to prepare a linear solution; Linear solution L6: Accurately measure 1 ml of the hydrazine sulfate stock solution prepared according to step (2c) of Example 1, place it in a 25 ml volumetric flask, dilute with water to the mark, shake well, take 1 ml, and follow the same procedure as in step "(2b) test solution" of Example 1, starting from "place in a 10 ml volumetric flask", to prepare a linear solution; The above six linear solutions were injected into a liquid chromatograph, and the chromatograms were recorded. The peak area, linear equation, slope, intercept, correlation coefficient, and response factor (peak area per unit concentration) of dibenzylhydrazine for each concentration solution were reported. The results are shown in the table below.

[0063] .

[0064] The above results show that the linear correlation coefficient of hydrazine is greater than 0.995, which is usually required in the art; the RSD of the response factor (peak area per unit concentration) at each concentration point is less than 15.0%, which is usually required in the art; and the absolute value of the Y-axis intercept is less than 10.0% of the peak area at the 100% limit concentration level, which is usually required in the art. This indicates that the method of Example 1 of the present invention has good linearity within the investigated concentration range.

[0065] The HPLC chromatograms of linear solutions L2 and L6 are shown below. Figure 5 and Figure 6 .

[0066] (4) Limit of Quantification and Limit of Detection Limit of Quantification Solution: Prepare 6 parallel solutions using the same method as linear solution L1 described above. Accurately measure 1 ml of each solution and follow the same procedure as in Example 1, "(2b) Test Solution", starting from "Place in 10 ml volumetric flask", to obtain 6 limit of quantification solutions.

[0067] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution and place it in a 10 ml volumetric flask. Dilute with water to the mark and shake well. Prepare 3 parallel portions. Accurately measure 1 ml of each portion and follow the same procedure as in Example 1, "(2b) Test solution", starting from "place in a 10 ml volumetric flask". Prepare 3 portions of detection limit solution.

[0068] Inject each of the above limit-of-quantitation (LOQ) solutions into the liquid chromatograph, record the chromatograms, and report the signal-to-noise ratio (S / N), concentration (ng / ml), equivalent concentration of the test sample solution (ppm), and peak area RSD of dibenzylhydrazine in each limit-of-detection (LOD) and limit-of-quantitation (LOD) solutions. The results of the limit-of-detection (LOD) test are shown in the table below.

[0069] .

[0070] The results of the limit of quantitation test are shown in the table below.

[0071] .

[0072] Conclusion: In the limit of detection solution, the peak of dibenzylhydrazine was 3≤S / N≤6; in the limit of quantitation solution, the peak of dibenzylhydrazine was 10≤S / N≤20, and the peak area RSD was less than 10.0% (n=6); indicating that the sensitivity of the method in Example 1 meets the detection requirements generally recognized by those skilled in the art.

[0073] (5) Accuracy Reference solution: prepared according to the method described in step (2e) of Example 1.

[0074] 50% limit concentration hydrazine sulfate solution: Accurately measure 1 ml of the hydrazine sulfate stock solution obtained by the method described in step (2c) of Example 1, place it in a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0075] 100% limit concentration hydrazine sulfate solution: Accurately measure 1 ml of the hydrazine sulfate stock solution obtained by the method described in step (2c) of Example 1, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0076] 150% limit concentration hydrazine sulfate solution: Accurately measure 3 ml of the hydrazine sulfate stock solution obtained by the method described in step (2c) of Example 1, place it in a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0077] Spiked test solution: Weigh approximately 25 mg of the test sample accurately and place it in a 10 ml volumetric flask. Accurately add 1 ml of hydrazine sulfate solution of each of the above limit concentrations. Follow the same procedure as in step (2b) of Example 1, starting with "shaking for 15 seconds", to prepare spiked test solutions of different concentrations. Prepare 3 copies of each concentration in parallel.

[0078] Inject the reference solution, 50% limit concentration spiked test solution, 100% limit concentration spiked test solution, and 150% limit concentration spiked test solution directly into the sample, record the chromatogram, and calculate the hydrazine content using the external standard method according to the following formula: , In the formula, m 对 The sample weight of the reference standard is in mg. m 空白样 The sample weight for the blank test sample is in mg. m 加样 The weight of the spiked test sample, in mg; C 对 The content of the reference standard is % . C 空白样 The content of hydrazine in the blank test sample, % V 加入 The volume of reference solution added to spike the test sample, in ml; A 加样 To determine the peak area of ​​dibenzylhydrazine in the spiked test solution; A 空白样 The peak area of ​​dibenzylhydrazine in the blank test solution; A 对 The peak area of ​​dibenzylhydrazine in the reference solution; S 对 This is the dilution factor of the reference standard; S 加样 This refers to the dilution factor of the spiked test solution; S 空白样 This represents the dilution factor of the blank test solution.

[0079] The results of the spiking accuracy test are shown in the table below: .

[0080] Conclusion: The recovery rate of hydrazine in the spiked test solution ranged from 93.9% to 106.4%, with an average recovery rate (n=9) of 101.4% and an RSD of 3.7%, which meets the generally accepted validation requirements in the art.

[0081] (6) Precision (repeatability and intermediate precision) Prepare the reference solution and the test solution according to the method described in Example 1, and prepare 6 copies of each in parallel for repeatability testing.

[0082] Different analysts (personnel 1 and personnel 2) used different instruments (Dionex Ultimate 3000 and Shimadzu LC-20AT) to prepare the reference solution and test solution according to the method in Example 1. Six copies of each solution were prepared in parallel for intermediate precision testing.

[0083] The reference solution and the test solution obtained from the above repeatability test and intermediate precision test were directly injected, the chromatograms were recorded, and the hydrazine content was calculated by the dibenzylhydrazine peak area according to the external standard method described in Example 1. The results are shown in the table below.

[0084] .

[0085] The average result of the 12 samples was 5.4 ppm, and the RSD of the 12 samples was 5.0%.

[0086] Conclusion: The hydrazine determination results in 6 repeatability test solutions were between 5.2 ppm and 6.1 ppm, with an RSD of 6.8%; the hydrazine determination results in 12 intermediate precision test solutions were between 5.1 ppm and 6.1 ppm, with an RSD of 5.0%, which meet the generally recognized validation requirements of those skilled in the art.

[0087] (7) Durability Blank solution, control solution and test solution were prepared according to the method of Example 1 for durability testing.

[0088] Take blank solution, reference solution and test solution, and inject them into the chromatographic conditions of Example 1 and modified chromatographic conditions (flow rate changed to 0.9 ml / min or 1.1 ml / min, or column temperature changed to 28 ℃ or 32 ℃). Record the chromatograms, examine the suitability of the system, calculate the hydrazine content in the test solution by external standard method, and examine the robustness of the analytical method. The results are shown in the table below.

[0089] .

[0090] The above results indicate that adjusting the main chromatographic parameters did not cause interference at the retention time of the dibenzylhydrazine peak with the blank solution; in the reference solution, the theoretical plate number of the dibenzylhydrazine peak was greater than 10,000; after six consecutive injections of the reference solution, the dibenzylhydrazine peak remained unchanged. The peak area RSD of hydrazine was less than 2.0%; the hydrazine concentration in the test solution was between 5.4 ppm and 6.1 ppm, indicating that the analytical method has excellent robustness.

[0091] The analytical method for determining the hydrazine content in carbidopa described in Example 1 of this invention was verified, and the results are summarized in the table below.

[0092] .

[0093] Example 3: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment is performed with reference to the operation described in "(2) Solution Stability" of Embodiment 1 and Embodiment 2. The only difference is the modification made to the operation in "(2b) Test Solution" of Embodiment 1, as follows.

[0094] Referring to Example 1, the 100 μl glacial acetic acid used in “(2b) test solution” was replaced with 50 mg benzoic acid (used in Cui’s literature, a 50% concentration solution prepared with methanol), and the rest of the operation remained unchanged. The stability test was conducted according to the description in "(2) Solution Stability" of Example 2. The results were as follows: The stability RD% of the hydrazine sulfate solution (free hydrazine 0.5 μg / ml) after being placed at room temperature for 24 hr and 48 hr before derivatization was -1.7% and 1.3% respectively, compared to 0 hr. The stability RD% of the reference solution (free hydrazine 0.05 μg / ml) after being placed at 10°C for 4 hr, 8 hr, 24 hr and 48 hr was 1.5%, 0.8%, -2.0% and 1.7% respectively, compared to 0 hr. The stability RD% of the test solution after being placed at 10°C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr and 2 hr was 2.4%, 5.8%, 12.4%, 15.7% and 17.6% respectively, compared to 0 hr, indicating that the RD% of the test solution exceeded 10% after 0.75 hr.

[0095] Example 4: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment is performed with reference to the operation described in "(2) Solution Stability" of Embodiment 1 and Embodiment 2. The only difference is the modification made to the operation in "(2b) Test Solution" of Embodiment 1, as follows.

[0096] Referring to Example 1, the 100 μl glacial acetic acid used in “(2b) test solution” is replaced with 100 mg benzoic acid (prepared with methanol to a concentration of 50%), and the rest of the operation remains unchanged. The stability test was conducted according to the description in "(2) Solution Stability" of Example 2. The results were as follows: The stability RD% of the hydrazine sulfate solution (free hydrazine 0.5 μg / ml) after being placed at room temperature for 24 hr and 48 hr before derivatization was 1.6% and 0.7% respectively, compared to 0 hr. The stability RD% of the reference solution (free hydrazine 0.05 μg / ml) after being placed at 10°C for 4 hr, 8 hr, 24 hr and 48 hr was -1.1%, 1.4%, -2.2% and -1.3% respectively, compared to 0 hr. The stability RD% of the test solution after being placed at 10°C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr and 2 hr was -2.1%, 4.4%, 13.7%, 16.4% and 17.3% respectively, showing that the stability RD% of the test solution exceeded 10% after 0.75 hr.

[0097] Example 5: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment is performed with reference to the operation described in "(2) Solution Stability" of Embodiment 1 and Embodiment 2. The only difference is the modification made to the operation in "(2b) Test Solution" of Embodiment 1, as follows.

[0098] Referring to Example 1, the 100 μl glacial acetic acid used in “(2b) test solution” was replaced with 150 mg benzoic acid (prepared with methanol to a concentration of 50%), and the rest of the operation remained unchanged. The stability test was conducted according to the description in "(2) Solution Stability" of Example 2. The results were as follows: The stability RD% of the hydrazine sulfate solution (free hydrazine 0.5 μg / ml) after being placed at room temperature for 24 hr and 48 hr before derivatization was 1.2% and -1.9% respectively, compared to 0 hr. The stability RD% of the reference solution (free hydrazine 0.05 μg / ml) after being placed at 10°C for 4 hr, 8 hr, 24 hr and 48 hr was 1.2%, 3.1%, -1.7% and -0.8% respectively, compared to 0 hr. The stability RD% of the test solution after being placed at 10°C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr and 2 hr was -0.4%, 5.1%, 10.3%, 14.6% and 15.8% respectively, showing that the stability RD% of the test solution exceeded 10% after 0.75 hr.

[0099] The results of Examples 3-5 above show that when benzoic acid is used instead of glacial acetic acid according to the approach in Cui's literature, the stability of the test solution deteriorates significantly.

[0100] Example 6: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment is performed with reference to the operation described in "(2) Solution Stability" of Embodiment 1 and Embodiment 2. The only difference is the modification made to the operation in "(2b) Test Solution" of Embodiment 1, as follows.

[0101] Referring to Example 1, the 100 μl glacial acetic acid used in “(2b) test solution” is replaced with 60 mg, 120 mg, or 180 mg benzenesulfonic acid (50% aqueous solution), and the rest of the operation remains unchanged.

[0102] The stability test was conducted according to “(2) Solution Stability” in Example 2. The results were as follows: Under all conditions, the stability RD% of the hydrazine sulfate solution after being placed at room temperature for 48 hours and then subjected to derivatization reaction was in the range of -1.7% to 2.5% compared to 0 hours; Under all conditions, the stability RD% of the reference solution after being placed at 10°C for 48 hours was in the range of -0.8% to 2.8% compared to 0 hours. The stability (RD%) of the test solution obtained by adding 60 mg of benzenesulfonic acid after standing at 10℃ for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr relative to 0 hr was 2.1%, 3.5%, 9.8%, 13.1%, 15.3%, and 17.4%, respectively. The stability (RD%) of the test solution obtained by adding 120 mg of benzenesulfonic acid after standing at 10℃ for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr relative to 0 hr was -1.5%, 4.7%, 11.2%, 13.7%, 15.1%, and 16.9%, respectively. The stability (RD%) of the test solution obtained by adding 180 mg of benzenesulfonic acid, after being placed at 10°C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr relative to 0 hr, was 1.1%, 3.9%, 10.9%, 12.8%, 14.8%, and 17.1%, respectively. This indicates that the RD% of the test solution exceeded 10% after 0.75 hr.

[0103] Example 7: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment is performed with reference to the operation described in "(2) Solution Stability" of Embodiment 1 and Embodiment 2. The only difference is the modification made to the operation in "(2b) Test Solution" of Embodiment 1, as follows.

[0104] Referring to Example 1, the 100 μl glacial acetic acid used in “(2b) test solution” was replaced with 50 µl, 100 µl, and 200 µl formic acid, respectively, while the rest of the operation remained unchanged. The stability test was carried out according to “(2) solution stability” in Example 2. The results showed that the stability RD% of the hydrazine sulfate solution after being placed at room temperature for 48 hours and then subjected to derivatization reaction was in the range of -1.5% to 2.1% compared to 0 hours under all conditions; the stability RD% of the reference solution after being placed at 10°C for 48 hours was in the range of -3.1% to 0.5% compared to 0 hours under all conditions. The stability (RD%) of the test solution obtained by adding 50 µl of formic acid after standing at 10 °C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr relative to 0 hr was 1.6%, 2.4%, 6.9%, 12.3%, 14.5%, and 16.7%, respectively. The stability (RD%) of the test solution obtained by adding 100 µl of formic acid after being placed at 10 °C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr, relative to 0 hr, was -1.1%, 2.8%, 7.3%, 11.2%, 15.7%, and 17.5%, respectively. The stability (RD%) of the test solution obtained by adding 200 µl of formic acid after being placed at 10 °C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr, relative to that at 0 hr, was 1.2%, -0.8%, 5.6%, 10.7%, 15.9%, and 16.5%, respectively. This shows that the RD% of the test solution exceeded 10% after 1 hr.

[0105] In this embodiment, the inventors also replaced glacial acetic acid with other acids, such as succinic acid, phosphoric acid, and hydrochloric acid, according to the methods of Examples 3-5. As a result, the stability of the test solution was significantly worse than that of glacial acetic acid. For example, after being placed at 10°C for 1 hour, the RD% of the solutions all exceeded 9%.

[0106] In fact, the inventors also unexpectedly discovered that the use of triethylamine in the preparation of the test solution is also necessary; otherwise, the stability of the aforementioned RD% will be significantly worse than in Example 1. In other words, the stability of the test solution is related to the combined use of glacial acetic acid and triethylamine, as shown in the following examples.

[0107] Example 8: Method for determining hydrazine in the active pharmaceutical ingredient carbidopa This embodiment is performed with reference to the operation described in "(2) Solution Stability" of Embodiment 1 and Embodiment 2. The only difference is that the method of using triethylamine is changed in the operation of "(2b) Test Solution" of Embodiment 1, as follows.

[0108] Referring to Example 1, the triethylamine used in "(2b) test solution" was changed to 0µl, 100µl, and 450µl, while the other operations remained unchanged. Stability tests were conducted according to "(2) solution stability" in Example 2. The results showed that the stability RD% of the hydrazine sulfate solution after being placed at room temperature for 48 hours before derivatization under all conditions was in the range of -2.3% to 1.8% compared to 0 hours; the stability RD% of the reference solution after being placed at 10°C for 48 hours under all conditions was in the range of -1.7% to 1.3% compared to 0 hours. The stability (RD%) of the test solution obtained by adding 0 µl of triethylamine after standing at 10 °C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, and 2 hr, relative to 0 hr, was 3.3%, 9.3%, 14.8%, 18.7%, 19.3%, and 18.5%, respectively. The stability (RD%) of the test solution obtained by adding 100 µl of triethylamine after being placed at 10 °C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 1.5 hr, 2 hr, and 3 hr relative to 0 hr was 1.7%, 4.7%, 7.6%, 9.4%, 12.7%, 15.2%, and 16.3%, respectively. The stability (RD%) of the test solution obtained by adding 450 µl of triethylamine after being placed at 10 °C for 0.25 hr, 0.5 hr, 0.75 hr, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, and 9 hr relative to 0 hr was 1.3%, -1.2%, -2.3%, -3.1%, 2.6%, 4.1%, 8.3%, 12.8%, and 16.6%, respectively.

[0109] In the additional test, the operation described in Example 1 was performed, except that glacial acetic acid was not added in the "(2b) test solution" operation of Example 1; the resulting test solution was tested according to the "(2) solution stability" of Example 2, and the RD% reached 6.8% after being placed at 10°C for 0.5 hr and 10.5% after being placed for 0.75 hr; the accuracy was tested according to the "(5) accuracy" of Example 2 (measured at 0.5 hr), and the average recovery rate for the low, medium and high addition amounts was 84.3% and the RSD was 4.4%; these results indicate that the stability does not meet the requirements when glacial acetic acid is not added, and the recovery rate is significantly lower and does not meet the determination requirements.

[0110] The above results show that the longer the test solution remains at a low RD% (stable) with increasing triethylamine content, the better it is for drug quality control. Those skilled in the art generally consider that the RD% value in the method of this invention is within ±10%, especially within ±5%, which meets the methodological requirements.

[0111] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for determining hydrazine in the active pharmaceutical ingredient carbidopa, comprising the following steps: pre-column derivatization-reversed-phase high-performance liquid chromatography. (1) Provide a chromatography system: including a high performance liquid chromatograph, a chromatographic column with octadecylsilane bonded silica gel as the packing material, a mobile phase of acetonitrile-water (70:30, v / v), and a detection wavelength of 305 nm; (2) Preparation of solution: (2a) Diluent: 2% benzaldehyde solution prepared using anhydrous methanol as solvent; (2b) Test solution: Weigh 25 mg of the carbidopa raw material to be tested accurately, place it in a 10 ml volumetric flask, add 1 ml of water accurately, shake for 15 seconds, immediately add 7 ml of diluent and 100 μl of glacial acetic acid accurately, shake for 60 seconds, immediately add 100 µl to 450 µl of triethylamine (preferably 300 µl), dilute to the mark with diluent, shake well, and the test solution is obtained; (2c) Hydrazine sulfate stock solution: Weigh 20.3 mg of hydrazine sulfate reference standard accurately, place it in a 200 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is ready; (2d) Hydrazine sulfate solution: Accurately measure 1 ml of hydrazine sulfate stock solution, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution; (2e) Reference solution: Accurately measure 1 ml of hydrazine sulfate solution and prepare the reference solution by following the same procedure as the test solution preparation method, starting from "placing it in a 10 ml volumetric flask". (2f) Blank solution: Accurately measure 1 ml of water and prepare it according to the same method as the test solution preparation method, starting from "place in a 10 ml volumetric flask" (equivalent to a free hydrazine concentration of 0); (3) System suitability test and requirements: After the liquid chromatography system has stabilized, inject a blank solution and record the chromatogram. The blank solution should not cause any interference at the retention time of the dibenzylhydrazine peak. Inject a reference solution and record the chromatogram. The system suitability test should meet the routine requirements. (4) Determination, limit judgment, and result calculation: Inject the reference solution and the test solution into the liquid chromatograph separately, and record the chromatograms; Limit: If there is a chromatographic peak in the chromatogram of the test sample solution with the same retention time as the dibenzylhydrazine peak, compare its peak area with the main peak area of ​​the reference solution. If the dibenzylhydrazine peak area in the chromatogram of the test sample solution is not greater than the main peak area of ​​the reference solution, it means that the limit of hydrazine content in the test sample is not greater than 20 ppm. The hydrazine content is calculated using the external standard method based on the peak area of ​​dibenzylhydrazine, in ppm. The formula is as follows: Hydrazine content = Sample peak area / Reference peak area × Reference concentration / Sample weight × Sample dilution factor × Conversion factor; In the formula, Conversion factor = Relative molecular mass of hydrazine / Molecular mass of hydrazine sulfate =32.0452 / 130.12 =0.2463。 2. The method according to claim 1, wherein the chromatographic column in step (1) is a Welch Xtimate C18 column.

3. The method according to claim 1, wherein the chromatographic column in step (1) is a Welch Xtimate C18 column with an inner diameter of 4.6 mm.

4. The method according to claim 1, wherein the chromatographic column in step (1) is a Welch Xtimate C18 column with a length of 250 mm.

5. The method according to claim 1, wherein the chromatographic column in step (1) is a Welch Xtimate C18 column with a packing particle size of 5 μm.

6. The method according to claim 1, wherein the flow rate of the mobile phase in step (1) is 1.0 ml / min.

7. The method according to claim 1, wherein the sample tray temperature in step (1) is 10°C; and / or the sample injection volume is 20 μl.

8. The method according to claim 1, wherein the test solution obtained in step (2b) is injected within 3 hours, for example, within 2 hours.

9. The method according to claim 1, wherein in step (3) system suitability test and requirements, the RSD of the dibenzylhydrazine peak area in the reference solution injected for 6 consecutive injections shall not be greater than 2.0%; and / or, the theoretical plate number of the dibenzylhydrazine peak in the chromatogram of the reference solution shall not be less than 5000.

10. The method according to claim 1, wherein in step (4), if the hydrazine content in the active pharmaceutical ingredient carbidopa is less than 20 ppm, the active pharmaceutical ingredient is considered to meet the requirements; for example, if it is less than 15 ppm, the active pharmaceutical ingredient is considered to meet the requirements; for example, if it is less than 10 ppm, the active pharmaceutical ingredient is considered to meet the requirements.

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